Isolation Culture And Identification Of Endothelial Progenitor Cells From Human Umbilical Cord Blood And Peripheral Blood

Injury of cerebral vessels is main pathological foundation of stroke,up to now, vascular anatomic structure are unble to completly recovery under drug treatment and interventional therapy, vascular disease will recurrence accompany with time lapsing. The classic paradigm for neovascularization is"angiogenesis", a process by which new vessels and new endothelial cells are derived from sprouting from preexisting differentiated endothelial cells. The identification of EPCs change our recognition of neovascularization,EPCs can be selectively recruitmented to damaged or ischemic tissue, and form new blood vessel in the place where no vessels exit before -vasculogenesis,which similar to primitive vascular net in embryonic period.Under the stress of anoxaemia condition, two ways of neovascularization coordinate with each other. The objection of this study is to isolate EPCs from umbilical cord blood (CB) and peripheral blood(PB),culture and identificate them so as to provide technical and theoretical support for the clinical application in cerebral ischemic stroke.Mononuclear cells were seperated using a Ficoll density gradient , isolated mononuclear cells were resuspended by M199 , consisting of 20% fetal bovine serum and VEGF, 1x106 mononuclear cells per well were seeded on fibronectin-coated 24-well plate and incubated in a 5%CO2 incubator at 37°C. The biological characteristics of attached cells were observedCD34,CD133 and CD105 as the specific markers of progenitor cells were detected by Flow Cytometry, KDR and CD31 as the endothelial cells markers were used for immunocytochemistry and immunofluorescence cytochemistry for the attach cells at day 7 of culture. To further verify that these cells were endothelial progenitor cells, uptaking of Dil-labelled acetylated LDL and binding of FITC-labelled UEA-1 were performed.The results are as follows:(1) The endothelial progenitor cells were isolated and obtained from PB and CB;(2) The biological characteristics of EPCs: a. grow at a delayed time course; b. blood island sample cell colony, an EPCs colony consists of a central cluster of rounded cells with surrounding radiating thin, flat cells c.head and tail connected as trabs and nets; d. cobblestone-like cells; this phenomenon was analogic with the primitive vascular net in embryogenesis, this similarity indicated that EPCs may contribute to postnatal angiogenesis;(3) The number of cell colony was significantly lower in PB than in CB;(4) Early EPCs and late EPCs had no significant differences in biologic behaviors;(5) Cells were positive for progenitor cell associated markers CD34,CD133 and CD105, the level are 74.4%,64%,83.1%and 60.4%,48.2%,42.9% respectively;(6) Cells cultured at day 7 were positive for CD34 and CD133 by immunocytochemistry;(7) Cells cultured at day 7 were positive for CD34 and CD133 by immnofluorescnce cytochemistry;(8) Cells could take up ac-LDL and bound UEA-1, this illustrated that the cells we obtained can oriented differentiated to EPCs.EPCs become a research hotspot till Asahara,et all. published the first detailed description of isolation of putative progenitor endothelial cells for angiogenesis in 1997. EPCs are pluripotent cells which exist in the fetal liver, cord blood and adult peripheral blood, and can be differentiated into endothelial cells. EPCs can repair damaged endothelia, attenuate the development of atherosclerosis and promote new vessels formation in ischemic tissue. Research showed that EPCs participate in angiogenesis after ischemic stroke, predict the development and prognosis of cerebral ischemia, fight against stent thrombosis and restenosis, and promote merve regeneration.EPCs were directly injected into the hindlimb ischemia animals model, ischemic hindlimb blood flow returned to normal amount of 69% at the first 28d, limb necrosis and amputation reduced by 50%, the proportion of limbs returned to normal up to 60% , which indicate that EPCs may contribute to angiogenesis, while angiogenesis provides the microvascular environment for nerve regeneration; Similarly EPCs were injected into the rabbit model of carotid artery denudation, the EPCs can speed up the reendothelialization of injuried artery, improve carotid artery intima-newborn, enhance vasodilation and secretion of vascular growth factors,which revealed that the EPCs can promote regeneration and repairment of damaged endothelium. The characteristics of EPCs provide theoretical support in the prevention of restenosis after stenting, EPCs also become an ideal cell source for building functional microvascular network.In recent years, EPCs transplantation gradually applied to the treatment of vascular-related diseases, EPCs as seed cells for gene therapy with stem cell therapy on the role of angiogenesis is significantly more effective.However,studies in cerebral ischemic stroke were deficient. The innovation of our experiment is based on the clinical point of view, select umbilical cord blood and peripheral blood which were easily accessible, search for a stable and practical method for isolation and identification of EPCs in vitro, and provide theoretical and technical support for EPCs autologous transfusion and the establishment of progenitor cell bank for the patients suffering from cerebral ischemic stroke.